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Chapter
The Role of Cell‑Free Rabbit
Reticulocyte Expression Systems
in Functional Proteomics
Michele Arduengo, Elaine Schenborn and Robin Hurst*
Abstract
I
n the broad area of functional proteomics, that is the global characterization of proteins and
their function, cell‑free rabbit reticulocyte lysate (RRL) has been used extensively to elucidate
the mechanisms of mammalian translation, cotranslational modifications, post‑translational
modifications and translocation of proteins. More recently, RRL has been used as the workhorse
for manufacturing the proteins engaged in interaction, selection and protein evolution studies
from DNA or mRNA libraries either in microarray, display or in vitro expression cloning (IVEC)
technologies. This chapter highlights recent functional proteomics applications that use cell‑free
mammalian RRL.
Abbreviations
CMM, canine microsomal membrane; ER, endoplasmic reticulum; ERAD, endoplasmic
reticulum‑associated degradation; GPI, glycosylphosphatidylinositol; IVC, in vitro compart‑
mentalization; IVEC, in vitro expression cloning; PCR, polymerase chain reaction; PTT, protein
truncation test; RRL, rabbit reticulocyte lysate; RT‑PCR, reverse transcription‑polymerase chain
reaction; SP cells, semipermeabilized cells.
HighWire Press is a registered trademark of Stanford University. TnT is a registered trademark
of Promega Corporation.
Introduction
In late 1950s and early 1960s researchers first demonstrated that radioactive amino acids could
be incorporated into hemoglobin in cell‑free rabbit reticulocyte lysate (RRL).1,2 Since then RRL
has been used to elucidate the highly complex events that encompass translation, from initiation
to termination.3‑5 RRL has also proven useful in understanding cotranslational folding of nascent
polypeptide chains, protein targeting and post‑translational folding. During the 1970s, researchers
showed that RRL could be manipulated for exogenously directed mRNA protein synthesis, so
that only a single protein of interest was synthesized.6 In the 1990s, the development of coupled
transcription/translation, in which RRL is supplemented with T7, T3, or SP6 RNA polymerases
further simplified the expression of protein targets.7,8 DNA‑directed protein synthesis in RRL
has some advantages over mRNA‑primed RRL including the elimination of mRNA handling,
and it usually achieves higher levels of protein synthesis. An advantage of cell‑free RRL over
other cell‑free systems (E. coli or wheat germ extracts) is that the mammalian environment more
*Corresponding Author: Robin
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Cell-Free Expression, edited by Wieslaw Kudlicki, Federico Katzen and Robert Bennett.
©2007 Landes Bioscience.
Cell-Free Expression
closely mimics human cells. Cell‑free RRL is generated from lysed reticulocytes isolated from
phenylhydrazine‑treated rabbits. The lysed reticulocytes are treated with microccocal nuclease
to remove endogenous mRNA. The RRL is optimized and supplemented with components that
give optimal translation when priming with mRNA and, in the case of a coupled system, optimal
translation/transcription for priming with DNA that contains the appropriate RNA phage poly‑
merase promoter sequences.
Cell‑free RRL has the same advantages as all cell‑free systems over cell‑based expression sys‑
tems: substantial time‑savings (two hours versus 24–48 hours for protein expression), the ability
to adapt to high‑throughput formats, increased tolerance to additives and less sensitivity to toxic
or proteolytic proteins. The current use of cell‑free RRL is substantial as illustrated by a HighWire
Press® search covering January 2000 to April 2006 that yielded more than 3,000 articles containing
the phrase “rabbit reticulocyte.” This chapter will focus on recent applications that use RRL for
cell‑free functional proteomics.
Membrane Topology
Synthesis of Membrane Proteins
Membrane protein topology is often described based on the predicted amino acid sequence and
algorithms that estimate hydrophobicity and probable secondary structure of a stretch of amino
acids.9 However, different algorithms or different stringencies applied to the same algorithm can
predict different topologies, and many algorithms fail to account for cotranslational processing
events or the effects of post‑translational modifications on protein topology.9 Translation in a
cell‑free system containing microsomes or semipermeabilized cells can provide empirical data
about membrane protein topology.
Using a prepared lysate supplemented with endoplasmic reticulum (ER)‑derived microsomal
membranes such as canine pancreatic microsomal membranes (CMMs)10‑12 or digitonin‑permea‑
bilized cells (semipermeabilized cells),13 membrane proteins can be successfully synthesized, trans‑
located and modified in vitro. Semipermeabilized (SP) cells have some advantages over ER‑derived
microsomes. SP cells are more likely to contain the necessary components for the correct folding
and modification of proteins normally expressed by the cells, and they have a spatially intact ER
and Golgi system that better approximates the cellular environment.13 Additionally, SP cells can
be more efficient at specialized modifications such as adding glycosylphosphatidylinositol (GPI)
anchors.14 Using SP cells and RRL without dithiothreitol allows disulfide bond formation and
efficient folding of proteins such as MHC class I heavy chain.15
Proteins can be associated with the membrane in a variety of ways. Integral membrane proteins
span both leaflets of the phospholipid bilayer with one or more alpha helical transmembrane do‑
mains consisting of approximately 20 hydrophobic amino acids. Peripheral membrane proteins
can be associated with a single membrane leaflet either by means of a fatty acid modification, such
as prenylation, myristoylation, or a GPI anchor, or by association of a predominantly hydrophobic
stretch of amino acids. Cell‑free translation systems such as RRL supplemented with microsomes
or SP cells can provide information about how a particular protein associates with a membrane. For
instance, treating isolated microsomes with sodium carbonate dissociates peripheral but not integral
membrane proteins from microsomes or semipermeabilized cells.16 Such treatment has been used
to show that the glycoprotein GP4 produced by the equine arteritis virus is an integral membrane
protein, while the GP3 protein, produced by the same virus, is membrane‑anchored.17
Protease Protection Assays
Protease protection assays are often used to help determine membrane protein topology and
orientation. Water‑soluble proteases cannot freely cross the lipid bilayer of microsomes, so segments
of proteins that are in the lumen of the microsomes will not be subject to protease digestion unless
the microsomes are first permeabilized. Assuming that the lumen of microsomes represents the
The Role of Cell-Free Rabbit Reticulocyte Expression Systems in Functional Proteomics
lumen of the ER, the segments of proteins in the lumen of the microsomes become extracellular
once a protein is inserted into the plasma membrane.
Several studies have used protease protection assays to determine the topology of membrane
proteins, including determining which of two proposed topologies is correct for cytochrome b5.18 In
this study, failure of carboxypeptidase Y to remove C‑terminal labeled methionines of cytochrome
b5 suggests that the C‑terminus is inside the lumen of the ER and inaccessible to the protease.18
Often, the protease assay is performed on proteins translated in the presence of microsomes or
SP cells. The microsomes are incubated with the protease with or without concomitant detergent
solubilization, and protease fragments are compared between the solubilized or unsolubilized
samples. Using a proteinase K protection assay of proteins translated in RRL supplemented with
CMMs, Umigai et al19 showed that the M2 domain of the K+ channel Kir 2.1 is oriented with the
C‑terminus toward the cytoplasm. A similar assay has been used to explore the effect of pathogenic
mutations on the prion (PrP) protein.14 In addition to determining topology of a particular protein,
researchers can use protease assays to dissect the process of ER binding and translocation.20
Tagging Membrane Proteins to Determine Orientation
Another strategy for determining membrane topology involves tagging a protein with an
enzyme or epitope. Tagging is often used in conjunction with other studies such as glycosylation
or protease assays to give a more complete picture of membrane topology. In one study, the
C‑terminal end of each of several deletion mutants of Presenilin I was tagged with E. coli leader
peptidase (LPase). Anti‑LPase antibody was able to immunoprecipitate in vitro translated protein
from some but not all of the deletion mutants based on the location of the C‑terminus of the
protein (either cytosolic or luminal).21 C‑terminal and N‑terminal glycosylation tags have also
been used in experiments to investigate the topology of vitamin K epoxide reductase.22
Glycosylation
N‑Linked Glycosylation of Membrane and Secreted Proteins
Glycosylation studies in RRL supplemented with CMMs or SP cells can provide information
about membrane topology. Portions of proteins translocated into the lumen of microsomes or
SP cells are exposed to enzymes responsible for core N‑linked glycosylation. N‑linked glyco‑
sylation acceptor sites can be inserted into the protein, at the N‑ or C‑terminus, for example.
Any sugar residues that are added should be removed by the actions of glycosidases, such as
endoglycosidase H, when microsomes containing the proteins are treated with detergents to
allow the glycosidase access to the luminal portion of the protein. Such a strategy was used to
determine the membrane topology of vitamin K epoxide reductase.22 Zhang and Ling used sensi‑
tivity to peptide N‑glycosidase (PNGaseF) to determine whether an 18 kDa protease‑protected
fragment from mouse P‑glycoprotein is glycosylated.23 Additionally, carrying out translation
in RRL supplemented with microsomes in the presence or absence of tunicamycin (a glycosyl‑
ation inhibitor) can allow comparison of glycosylated and unglycosylated forms of proteins
produced in vitro.24
The membrane topology of polytopic proteins (proteins that span the membrane multiple
times) can be especially difficult to predict. The Presenilin‑1 protein is a polytopic protein pre‑
dicted by hydrophobicity analysis to span the membrane from six to eight times. To determine
membrane topology for Presenilin‑1, a series of C‑terminal deletions was made to remove predicted
transmembrane regions of the protein. The truncated proteins were translated in vitro in the
presence of microsomes. Endoglycosidase H sensitivity of protein from solubilized microsomes
changed as deletions of the transmembrane domains altered the orientation of the protein in the
membrane.21 Combined with protease protection assays and epitope tag labeling at the C‑terminus,
these glycosylation results supported a seven‑transmembrane domain structure with an additional
membrane‑embedded domain for Presenilin‑1.
Cell-Free Expression
O‑linked Glycosylation of Cytosolic and Nuclear Proteins
Secretory and membrane proteins are not the only proteins in the cell that are glycosylated.
Many nuclear and cytosolic proteins are modified by O‑linked glycosylation (O‑GlcNAcylation).25
RRL contains the enzymes and substrates necessary for the O‑GlcNAcylation of proteins,26 and
addition of microsomes or SP cells provides the environment necessary for correct membrane‑
protein folding. To assess whether the insulin‑responsive glucose transporter GLUT4 undergoes
O‑GlcNAcylation, GLUT4 cDNA was transcribed and translated in RRL supplemented with
CMMs.25 RRL was able to successfully modify GLUT4 protein.25
Lipid Modification and Acetylation of Proteins
Glycosylphosphatidyl Inositol (GPI) Anchors
Some proteins are anchored to the cell membrane by means of a glycosylphosphatidyl inositol
(GPI) modification at the C‑terminus.27 Unlike proteins incorporated into the membrane by a
transmembrane domain, proteins that are GPI‑anchored are reversibly associated with the lipid
bilayer. GPI anchoring does not appear to be necessary for cell survival, but it is necessary for
development.28 Proteins that are modified by the addition of a GPI anchor contain two signal
sequences, one at the N‑terminus that directs protein synthesis to the ER and a second at the
C‑terminus that directs the addition of the GPI‑anchor by a transamidase activity.29 Small nucleo‑
philic compounds like hydrazine can substitute for GPI, providing a means to assess whether GPI
modification has occurred by comparing the molecular weight of proteins translated in the presence
or absence of hydrazine.30 Human placental alkaline phosphatase (PLAP) is a GPI‑anchored pro‑
tein.24 GPI‑anchored mini‑PLAP has been generated by numerous groups using nuclease‑treated
RRL supplemented with microsomal membranes from CHO, F9, EL4 or K562 cells,24,28,29,31‑33
demonstrating that GPI modification can be reconstituted in a cell‑free system.
Prenylation
Some proteins are modified by prenylation, the attachment of one or more isoprenoid groups,
such as the 15‑carbon farnesyl group or the 20‑carbon geranylgeranyl group, to a cysteine residue.
Prenylation can mediate membrane association of some proteins, particularly the Ras‑like GTPases,
and protein‑protein interactions (e.g., nuclear lamins).34 Prenylated proteins can be produced
and detected in RRL supplemented with the labeled isoprenoid precursor mevalonic acid after
the translation reaction is complete; additionally proteins synthesized in RRL can be modified
using photoactivatable analogs of isoprenoids.35‑37 Gel‑based assays to detect changes in protein
migration as a result of prenylation are also used, but these are indirect assays and are usually
performed along with a labeling experiment. Most prenylation assays require autoradiography
of the labeled lysate, which can take weeks or months. Benetka and colleagues have developed
an in vitro prenylation assay using N‑terminal GST‑tagged proteins and detection of 3H‑labeled
precursors using a TLC linear analyzer.38 The incorporation of the GST tag allows the labeled
protein of interest to be separated from free radioactive label and other proteins in the RRL, and
using the TLC scanner to detect the incorporated lipid molecule significantly reduces the time
required to obtain results.
N‑Myristoylation and Palmitoylation
Many proteins in eukaryotic cells are subject to N‑myristoylation, the addition of a 14‑
carbon fatty acid to the N‑terminus.39 In addition to being prenylated, the alpha subunits of
some G‑proteins, including pp60src and p21ras, are myristoylated. Other G‑protein alpha subunits,
including some of the Gs and Gq subunits, are not myristoylated, but are instead modified by the
attachment of a 16‑carbon palmitic acid (palmitoylation), and others are both myristoylated and
palmitoylated. Some studies suggest that N‑myristolyation and palmitoylation contribute to the
membrane association of these proteins.40‑42 N‑myristoylation can occur cotranslationally, imme‑
diately after the removal of the N‑terminal methionine from a protein or even post‑translationally
as with the protein BID (BCL‑2 interacting domain), a substrate of caspase‑8.39,43 Upon cleavage
The Role of Cell-Free Rabbit Reticulocyte Expression Systems in Functional Proteomics
by caspase‑8, BID reveals an N‑myristoylation site. RRL contains the components necessary
to complete N‑myristoylation, and has even been used to myristoylate tumor necrosis factor, a
normally nonmyristoylated protein, when it is modified to contain the N‑myristolyation motif
of other myristoylated proteins.44,45 The Arabidopsis SOS3 protein involved in plant salt tolerance
has also been synthesized and myristoylated in an RRL system.46
N‑Acetylation
A majority (70–85%) of the proteins found in the cytoplasm or nucleus of eukaryotes may be
modified by N‑acetylation.47,48 Examples of N‑acetylated proteins include ovalbumin, actin and
cytochrome c. Acetylation is catalyzed by N‑acetyltransferases cotranslationally after the initiator
methionine has been cleaved.48 Many proteins are also acetylated post‑translationally at internal
sites by acetyltransferase enzymes different from those involved in cotranslational modification.48
Acetylation of modified tumor necrosis factor (TNF) has been demonstrated in RRL,47 and acety‑
lation in RRL can be inhibited by the use of S‑acetonyl‑CoA, an analog of acetyl‑CoA.49
Reconstituting ER‑Associated Protein Degradation
Conditions such as environmental stress, viral infection and the absence of required partner
proteins can result in the accumulation of aberrantly folded proteins in the rough endoplasmic
reticulum (RER). The RER has a “quality control” system that targets these misfolded proteins
for degradation. This process, ER‑Associated Degradation (ERAD), requires ATP and is distinct
from the lysosomal degradation pathway in cells.50
RRL in conjunction with CMMs or SP cells has been used to reconstitute ERAD activity.15, 51
RRL has several advantages over intact‑cell systems for such study. The protein of interest will be
the only protein labeled in the RRL, making its degradation easy to follow.51 Second, a variety
of microsomal membranes can be used with RRL to reconstitute the activity.15,51 Because hemin
inhibits the proteasome activity of ERAD, degradation studies are best performed in RRL that
does not contain exogenous hemin. Researchers have reported that RRL that works well for studies
of degradation is usually poor for translation.51 Additionally, since ERAD is ATP‑dependent, the
RRL will need to be supplemented with ATP and an ATP regeneration system,51 and some authors
report that excess unlabeled methionine seems to aid in reconstituting ERAD activity.52
RRL‑based protein degradation systems have been used to investigate the synthesis, stability
and degradation of a variety of wild type and mutant proteins. In one such study, tyrosinase
ERAD was reconstituted in a commercially available RRL system supplemented with an ATP
regeneration system.53 Wild type and mutant tyrosinase associated with albinism were translated
in the presence of SP melanocytes; the SP cells were isolated and then resuspended in RRL
with the ATP regeneration system. Both proteins were degraded, although the mutant protein
degraded at a higher rate than the wild type protein. RRL‑based and rat cytosol‑based degra‑
dation systems have also been used to investigate the degradation of Apoprotein B (apoB).54
Aliquots of the transcription/translation reaction of HA‑tagged apoB were incubated in the
presence of an ATP regeneration system in fresh RRL or rat hepatocyte cytosol with or without
proteasome inhibitors. Inhibition of apoB degradation was more obvious in the rat hepatocyte
cytosol, presumably because RRL contains factors that interfere with the proteasome inhibitors.
To assess the role of the chaperone protein, hsp90, in the degradation of apoB48, geldanamycin
(GA), an antibiotic that competes for the ATP binding site on hsp90, was added to pelleted
CMMs before the degradation assay. There was no significant decrease in the amount of apoB48
in the presence of GA, indicating that GA did inhibit degradation and hsp90 was required for
apoB48 degradation.54
RRL has been used to reconstitute the degradation of α1‑antitrypsin Z [(α1AT)Z].52 Individuals
who are homozygous recessive for a mutation resulting in a Glu342 to Lys substitution have increased
susceptibility to liver disease.52 The amino acid substitution disrupts proper folding of (α1AT)Z,
and individuals susceptible to the liver disease are not able to degrade the misfolded protein effi‑
ciently. Mutant and wild type (α1AT)Z degradation were examined using an RRL degradation assay
system. The mutant (α1AT)Z was degraded efficiently. Mutant protein produced in the presence
Cell-Free Expression
of salt‑washed or puromycin‑treated, salt‑washed microsomes was also degraded, indicating that
the full complement of RER proteins was not required for degradation.52
Several mechanisms have been suggested to control the targeting of proteins accumulated in
the ER for degradation, including regulating the trimming of N‑linked oligosaccharide chains.
Oligosaccharide side chains can be modified by mannosidase I in the ER. Inhibiting this activity
seems to stabilize misfolded proteins.15 Wild type MHC class I heavy chain and a mutant heavy
chain that lacks the N‑linked glycosylation site but that can assemble into functional MHC class I
molecules were translated in RRL in the presence of SP HT1080 cells.15 The wild type protein was
degraded more quickly than the mutant, indicating that glycosylation is important for ERAD.
In Vitro Viral Assembly
The ability to reconstitute cotranslational assembly events and protein interactions using
cell‑free RRL systems can be extended to the study of in vitro mammalian viral protein assembly,
viral protein interactions with other cellular components, and viral protein effects on translation.
Early studies of viral protein assembly demonstrated that capsid proteins expressed in RRL were
capable of self‑assembling in vitro. For example, adenovirus type 2 fiber protein synthesized in
RRL formed trimers without requiring additional viral proteins or components.55 From this
relatively well‑defined, single‑protein model, more complex viral protein interactions and as‑
sembly studies evolved. Human papillomavirus‑like particles have been assembled in vitro from
L1 capsid protein expression in RRL.56 These particles also mimicked endogenous virus in its
conformational epitope exposure, and antibodies generated against the in vitro‑assembled L1
particles were effective in recognizing similar epitopes in patient samples. In addition to human
papillomavirus, human hepatitis C (HCV) core viral capsid precursor structures were also gen‑
erated de novo in reticulocyte lysates.57 Cell‑free systems have allowed detailed examination of
HCV core capsid assembly processes and properties, whereas mammalian culture systems have
been limited by low viral titers.
In vitro expression of Gag precursor proteins in RRL has allowed detailed examination of the
more complex pathway for retroviral assembly, which includes not only protein interactions, but
also plasma membrane interactions and budding. Viral capsid structures of immunodeficiency
virus type 1 (HIV‑1) have been assembled from the Gag precursor protein p55gag expressed in
RRL, and these particles resembled immature HIV‑1 viral structures.58 Additional processing of
proteins in viral capsids can include prenylation and glycosylation. Myristoylation of HIV‑1 viral
particles59 and glycosylation of woodchuck hepatitis virus capsid proteins60 have been investigated
using cell‑free RRL systems. These results illustrate the importance of cell‑free protein expression
in delineating processes involved in viral particle assembly pathways.
Protein Microarray Technology
In the field of functional proteomics, protein microarrays are filling a niche for miniaturization
coupled with high‑throughput assay capability.61,62 Protein microarray concepts are patterned after
DNA microarrays, but immobilization of diverse types of proteins in a manner that preserves con‑
formation and functionality is a complex and challenging problem to solve. Continuing advances
in microarray surface chemistries coupled with improvements in protein production capabilities,
sensitive detection methods, and instrumentation are accelerating the pace of development for
protein microarrays. Microarray formats include printing proteins at high density on a glass slides
or other solid surfaces, using miniaturized reaction chambers adapted for slides, and assaying
samples in multiwell plates.
Currently two general types of protein microarray applications are being pursued: antibody‑ or
peptide‑based arrays and functional protein arrays. Antibody‑ or peptide‑based arrays bind pro‑
teins of interest in given samples, such as serum, and can provide information about the amount
and specificity for binding of such proteins. This is referred to as protein profiling. The search
for disease biomarkers for diagnostic purposes and drug screening capabilities drives many of
the innovations for these types of arrays. Another strategy is to use protein microarray formats
The Role of Cell-Free Rabbit Reticulocyte Expression Systems in Functional Proteomics
Figure 1. Schematic of multiwell format protein array experiment. Tagged proteins are generated through coupled translation and then bind to coated multiwell surface. Printed with
permission from Promega Corporation.
to interrogate and carefully perturb protein functions such as protein‑protein, protein‑nucleic
acid or protein‑small molecule interactions, as well as protein enzyme activities. Cell‑free protein
expression systems, such as RRL, are well suited to supply the functional proteins required for
these types of protein microarrays. Cell‑free protein expression in RRL is versatile and allows
incorporation of specific moieties into the protein sequence for immobilization or detection
strategies, as well as post‑translational modifications, in an automatable manner.
Early proof‑of‑principle for use of RRL in combination with a multiwell array‑type format was
demonstrated by He and Taussig in 2001,63,64 and named “PISA” (Protein In Situ Array). Proteins
with double (His)6 tags were expressed directly from DNA templates with RRL in each well, and
the expressed proteins were immobilized by the (His)6 tag to nickel‑coated surfaces. Expression
and immobilization of functional proteins were demonstrated by enzymatic activity of a cloned
(His)6 tagged‑luciferase and a tagged‑single chain antibody fusion that bound its corresponding
antigen, progesterone. Microwells offer an advantage of maintaining aqueous reaction conditions
that are compatible with native protein conformation, compared to more harsh conditions pres‑
ent on a spotted glass slide. Expression of tagged protein expressed in cell‑free extracts eliminates
laborious protein purification schemes (Fig. 1).
Cell-Free Expression
Oleinikov et al65 describe a variation on protein arrays based on expression of protein from
RRL with concomitant immobilization. This strategy exploits features of electronic semicon‑
ductor microchips for protein binding and detection. A second variation of protein arrays takes
advantage of the stability of immobilized DNA in microarrays, protein expression and spatially
defined protein capture.66 This approach, named NAPPA (nucleic acid programmable protein
array) involves generating protein in situ with a coupled transcription/translation RRL layered
onto slides printed with a mixture of biotinylated DNA, avidin and polyclonal GST antibody.
Target proteins are expressed from template DNA encoding a C‑terminal GST fusion tag and
then the fusion proteins are spatially bound to the GST antibody. The target proteins are detected
using a monoclonal antibody to GST.
Protein Interaction with Other Molecules
Cell‑free RRL has been used to examine numerous protein‑protein,70 protein‑DNA,71,72 and
protein‑RNA interactions73‑75 via immunoprecitation67,74‑76 and tagged protein pull‑down.68,70,72
Confirmation of protein‑protein interactions in vitro often includes expression of target proteins
in RRL and pull‑down with a tagged protein, such as GST‑tagged protein, which is bound to
a solid support. These types of GST‑pull‑down experiments have been used to confirm spe‑
cific interactions of protein involved in signal transduction,77,78 transcription regulation,79‑81 ion
channels82 and splicesosomes.83 Reconstitution studies can be performed in which expressed pro‑
teins form a complex in cell‑free RRL and give a measurable biochemical response that mimics
an in vivo response. One interesting recent example is the demonstration that p43, a telomerase
accessory protein, can affect the in vitro nucleotide addition activity and processivity of the con‑
served core consisting of the protein telomerase reverse transcriptase (TERT) and the telomerase
RNA subunit. The resulting reconstituted ternary complex [TERT•RNA•p43] was identified and
examined by immunoprecipitation in coupled transcription/translation in RRL.76
Another interesting example of protein‑RNA interaction in RRL involved the depletion
of the internal ribosome entry site (IRES)‑interacting protein of the RRL by the immobilized
foot‑and‑mouth virus (FMDV)‑IRES.84 The effect of the depleted lysate was assessed by transla‑
tion efficiency of transcripts that were either capped or had FMDV IRES in the sense or antisense
orientation. This procedure should be useful for analysis of protein‑RNA interactions and their
role in IRES‑dependent translation.
Cell‑free translations in RRL can also provide information about protein‑protein interactions.
Translation in RRL with microsomal membranes and immunoprecipitations have helped to
elucidate the mechanism of activation of the endogenous p21‑activated kinase 2 (PAK‑2) by Nef
proteins that are encoded by human immunodeficiency virus (HIV) and simian immunodeficiency
virus (SIV) in vitro.67 The cell‑free system allows further investigation of the molecular mechanism
of activation because the timing and order of component additions can be easily controlled.
Protein interactions that involve ribosome‑associated chaperones (cotranslational folding
and targeting) and post‑translational interactions have also been explored using RRL.68‑70 During
cotranslational folding the nascent chains interact with chaperones such as the 70‑kD heat shock
protein cognate (Hsc‑70) and nascent polypeptide‑associated complex (NAC)68,85 and chapero‑
nins such as the Tailless complex polypeptide 1 (TCP1) ring complex (TRiC).61,87 Recently the
interaction of the TRiC with the nascent polypeptide chain as it emerges from the ribosome was
demonstrated using photoreactive Nε‑(5‑azido‑2‑nitrobenzoyl)‑Lys‑tRNAlys along with translation
of truncated actin in the RRL. Post‑translation interactions of chaperones have been investigated
using mutagenesis and immunoprecipitation from RRL after the expression of protein kinases.
These studies showed that phosphorylation of Ser12 of the Hsp‑90 cochaperone Cdc37 is critical
for its interaction with eukaryotic protein kinases and Hsp‑90.70
Historically, protein‑DNA interactions have been identified via mobility shift assays71,72 in
which the DNA binding activity of proteins expressed in RRL are visualized by a shift of molecular
weight on native polyacrylamide gels. Human biliverdin reductase (hBVR) is a serine/threonine
kinase that catalyzes the reduction of biliverdin to bilirubin in response to oxidative stress. Using
The Role of Cell-Free Rabbit Reticulocyte Expression Systems in Functional Proteomics
hBVR and hBVR mutants that were translated in RRL and analyzed using a mobility shift assay,
hBVR was found to bind to specific DNA sequences.71
Display Technologies
Cell‑free display technologies, such as ribosome display,86‑91 mRNA display92‑94 or in vitro virus
(IVV),95 and in vitro compartmentalization (IVC)96 are powerful technologies that can be used
to identify protein‑target molecule interactions and for directed evolution of proteins for desired
improvements. These technologies rely on coupled transcription/translation or translation using
RRL or other sources of lysates. Cell‑free display technologies have advantages over cell‑based
display technologies such as phage display,97 and cell surface display on bacteria98 or yeast.99 The
cell‑based display systems have limited library diversity because of transfection inefficiencies, the
inability to specify incorporation of nonnatural amino acids via amber suppressor tRNAs and bias
against cytotoxic proteins. Display technologies rely on coupling genotype (mRNA) to phenotype
(protein) to retrieve the genetic information along with protein function.
Eukaryotic Ribosome Display
Eukaryotic ribosome display is an entirely cell‑free technology that screens and selects
functional proteins and peptides from large libraries. For ribosome display, the link between
genotype and phenotype is accomplished by an mRNA‑ribosome‑protein (PRM) complex that
is stable under controlled conditions. The eukaryotic method of ribosome display using RRL for
coupled transcription/translation has been used to display single‑chain antibodies to form an
antibody‑ribosome‑mRNA (ARM) complex.87,88,91 The function of the single‑chain antibody is
evaluated by its binding properties to an immobilized antigen. The function of other non‑antibody
proteins can be evaluated by using a different immobilized target, such as a partner protein, ligand
or substrate, to capture the relevant PRM complexes. The mRNA that is complexed with the
protein can then be amplified by reverse transcription polymerase chain reaction (RT‑PCR) and
recovered as DNA. If screening and selection is the goal, then proofreading DNA polymerases
are necessary; however, if evolution or diversification of the DNA sequence pool is required, then
a nonproofreading polymerase such as Taq DNA polymerase is used. The major distinguishing
feature between eukaryotic ribosome display and prokaryotic ribosome display86,90 is that in
eukaryotic ribosome display, the RT‑PCR is carried out on the intact PRM complexes rather than
on mRNA that has been released from PRM complex.
Eukaryotic ribosome display has been used to select the enzyme, sialyltransferase II, from
a cDNA library in a 96‑well plate coated with the substrate, ganglioside GM3.100 Coupled
transcription/translation in an RRL expression system from the cDNA library resulted in an
enzyme‑specific protein‑ribosome‑mRNA (PRIME) complex. A recently described modification
of eukaryotic ribosome display incorporates Qβ RNA‑dependent RNA polymerase into the display
and selection process.101 This allows a continuous in vitro evolution (Fig. 2). The cell‑free RRL
is used in the coupled transcription/translation mode to generate mRNA and then protein. The
Qβ RNA‑dependent RNA polymerase mutates the generated mRNA and thus the simultaneous
display of the protein generated from the original mRNA. The ribosome ternary complexes display
the synthesized proteins/single chain antibodies and are selected against immobilized antigens.
For the selection process, the displayed wild type and mutants are competing for the target. The
recovery of the mRNA is the same as in ARM display.
Recently, improvements have been developed for eukaryotic ribosome display that allow
20.8‑fold more efficient selection102 than current methods, making ribosome display a readily
accessible technique for all researchers.
mRNA Display or in Vitro Virus
A different approach to the selection and identification of functional proteins is mRNA dis‑
play, also called in vitro virus,95 a technique that uses the cell‑free RRL translation system to link
a peptide or protein covalently to its encoding mRNA.92‑94 The mRNA has a puromycin‑tagged
DNA linker ligated or photo‑crosslinked to the 3´‑terminus.103 The ribosome will stall when
10
Cell-Free Expression
Figure 2. The proposed continuous in vitro evolution (CIVE) cycle. The coupled transcription/translation system with inclusion of Qβ replicase. This produces a process in which, in the reaction
mix, mRNAs are transcribed from the DNA template by T7 polymerase, replicated and mutated
by the Qβ replicase, translated and displayed on the surface of the ribosomes. In selecting against
a target, the displayed wild type and mutant are competing for the target. Reprinted from: Irving
RA et al, J Immunol Meth 248:31-45; ©2001 with permission from Elsevier.101
it reaches the mRNA‑DNA junction, and puromycin enters the ribosomal A‑site. The nascent
peptide is coupled to the puromycin by the peptidyl‑transferase. The complex of peptide‑
puromycin‑DNA linker‑mRNA is dissociated from the ribosome and can then be used for selec‑
tion and identification of functional protein (Fig. 3). mRNA display or IVV in combination with
in vitro selection are powerful tools for evolving and discovering new functional proteins. mRNA
display of a random peptide library has been used to determine the epitope‑like consensus motifs
that define the determinants for binding of the anti‑c‑Myc antibody.104 Recently, a method has
been described for mRNA display using a unidirectional nested deletion library.105 The method
identified high‑affinity, epitope‑like peptides for an anti‑polyhistidine monoclonal antibody and
should be useful for determining minimal binding domains and novel protein‑protein interactions.
Also, mRNA display can be used to select mRNA templates capable of efficiently incorporating
the nonnatural amino acid, biotinyl‑lysine, a lysine derivatized at the epsilon amino via amide
linkage to biotin.106 To generate the mRNA‑peptide fusions, the template library and the tRNA
pools are added to RRL for translation. The mRNA‑peptide fusions contain a mixture of peptides,
some of which contain the biotinyl‑lysine. Those that contain biotinyl‑lysine can be purified by
binding to streptavidin‑agarose.
In Vitro Compartmentalization
In vitro compartmentalization (IVC) links genotype and phenotype by compartmentalization
into discrete water‑in‑oil emulsions.96 Until recently, IVC has mainly been used in conjunction
The Role of Cell-Free Rabbit Reticulocyte Expression Systems in Functional Proteomics
11
Figure 3. Protein:RNA fusion. Covalent RNA:protein complexes can be generated by ligation of a DNA:puromycin linker to the in vitro transcribed mRNA. The ribosome stalls at the
RNA:DNA junction. Puromycin then binds to the ribosomal A site. The nascent polypeptide
is thereby transferred to puromycin. The resulting covalently linked complex can be used
for selection experiments. Reprinted from: Schaffitzel C et al, J Immunol Meth 231:119-135;
©1999 with permission from Elsevier.90
with prokaryotic coupled transcription and translation (E. coli S30 extract) for selection of peptide
ligands107‑109 and directed evolution of Taq DNA polymerase,110 bacterial phosphotriesterase111 and
DNA methyltransferase.112 However, IVC has been used in conjunction with coupled transcrip‑
tion/translation in RRL to select active restriction enzymes from a randomized large (109–1010
molecules) mutant Fok I library.103 Use of RRL in this way is made possible by a new inert emulsion
formulation that is compatible with coupled transcription/translation,114 allowing an expanded
range of vertebrate protein targets that may be difficult to express as soluble and functional in S30
(bacterial) or wheat germ lysates.
Screening
In Vitro Expression Cloning (IVEC)
IVEC115,116 is another approach that uses RRL for coupled transcription/translation to identify
genes and elucidate protein interactions with other molecules. Using this method cDNAs or small
plasmid pools (50–100 clones) are expressed and then assayed for a specific function. Plasmids from
the positive pools are further deconvoluted and rescreened (Fig. 4). The process is repeated until a
single positive plasmid has been identified. IVEC is only successful if the specific function assayed
can be distinguished from the endogenous activity in cell‑free RRL. IVEC has been successfully
used to identify protein substrates,117‑120 protein‑protein interactions,121,122 enzymatic activity,123‑125
protein‑DNA interactions,126 and phospholipid‑protein interactions.136
12
Cell-Free Expression
Figure 4. The strategy of in vitro expression cloning. An unamplified cDNA expression library
is plated at a density of approximately 100 clones per bacterial plate. Pooled plasmid DNA
is obtained by scraping colonies from each plate and performing a small-scale plasmid
purification. Each plasmid pool is then transcribed and translated in vitro with a commercially
available system, such as the TnT® Coupled Transcription/Translation Systems from Promega
Corporation. The resulting protein pool is then assayed for the presence of an activity. In
the illustrated experiment, a radioactive amino acid is included in the translation system to
specifically label the pool of proteins. Incubation of a pool with a modifying enzyme (lanes
labeled +) such as a protease or kinase can result in a change in mobility of a substrate (bands
marked with asterisk). Pool 1 contains a protein whose mobility is reduced following treatment
with a kinase; Pool 2 contains a protein that is degraded following treatment with an extract
containing an activated proteolytic system; Pool 3 contains a protein that is specifically cleaved
following treatment with a protease, decreasing its apparent molecular mass. Once a pool
containing a candidate activity is identified, the original cDNA pool is subdivided and retested
until the single cDNA encoding the protein of interest is isolated. Reprinted with permission
from: King RW et al, Science 277:973; ©1997 AAAS.115
The Role of Cell-Free Rabbit Reticulocyte Expression Systems in Functional Proteomics
13
Protein Truncation Test
The protein truncation test (PTT) is a mutation detection technique that specifically identifies
pathogenic premature termination codons and has the advantage of not detecting polymorphisms.
Using extracted RNA, the coding region is screened for truncated mutations. The RNA is subjected
to RT‑PCR such that the cDNA product contains a T7 promoter. The cDNA is subjected to
coupled transcription/translation in cell‑free RRL, and the translated products are analyzed on gels
to identify the truncated proteins. The PTT has been applied to screening for many clinical condi‑
tions including hereditary breast and ovarian cancer (BRCA I and BRCA II),128 colorectal cancer
(APC),129 Duchenne Muscular Dystrophy (DMD),130 and neurofibromatosis type I (NFI).131
Other Screening
An approach to screening that uses cell‑free RRL and site‑directed mutagenesis to identify
elements critical for protein N‑myristoylation132 has recently been described. Sequential vertical‑
scanning mutagenesis in the N‑terminal region of tumor necrosis factor (TNF), followed by
cotranslation N‑myristoylation in RRL revealed the major sequence requirements for protein
N‑myristoylation. RRL has also been used to functionally screen a randomly mutagenized
phage library.133 In this example, critical amino acids in the protein C1 were identified that were
responsible for binding RNA. Other amino acids were identified that were important for protein
oligomerization. Protein C1 is a member of the heterogeneous ribonucleoproteins that bind
nascent RNA transcripts.
Screens that use cell‑free RRL have also been developed to identify small‑molecule inhibitors
of translation.134 Numerous reports have implicated alternative translation initiation controls
occurring in cancer cells,135‑139 and small‑molecule inhibitors may provide tools for determining
the molecular mechanism of this alternative regulation. A high‑throughput screen was designed
to identify small‑molecule inhibitors of eukaryotic translation as well as inhibitors that interact at
the mRNA‑ribosome level to inhibit gene‑specific translation. This multiplex in vitro translation
was used to screen over 900,000 distinct compounds identified novel inhibitors.134 A secondary
high‑throughput eukaryotic translation screen to discover broad spectrum antibacterial com‑
pounds has also been used to assess the biochemical selectivity of the compounds for prokaryotic
translation.140
Another screen using RRL is a PCR‑based rapid detection screen for pyrazinamide
(PZA)‑resistant Mycobacterium tuberculosis.141 After amplification of the pncA gene and coupled
transcription/translation of the PCR product, the activity of the enzyme pyrazinamidase is
measured by the conversion of PZA to pyrazinoic acid. Other PCR‑based coupled transcrip‑
tion/translation methods for rapid phenotypic screening have also been developed, including
screening of the thymidine kinase gene for monitoring acyclovir resistant herpes simplex virus
and varicella‑zoster virus.142
Conclusions
Cell‑free RRL plays an important role in functional proteomics, whether the protein is destined
for the ER, modification, degradation, or forms a complex with DNA, RNA and other proteins.
RRL provides a mammalian environment for elucidating quasi‑cellular mechanisms and can be
easily manipulated by depleting or adding protein, tRNA or membranes to provide the desired
environment so that the function of a protein or proteins can be studied. Additionally, cell‑free
RRL is proving to be a useful tool for high‑throughput protein synthesis in protein microarrays
and other screening situations.
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